# Patent

## Title
Corneodesmosin based test and model for inflammatory disease

## Abstract

The present invention relates to a polynucleotide encoding the corneodesmosin protein having one or more nucleotide insertions, deletions or substitutions at one or novel positions. The invention also relates to the corneodesmosin protein having one or more amino acid insertions, deletions and substitutions. These nucleotide and amino acid polymorphisms are useful in diagnosing or determining susceptibility to corneodesmosin-mediated disease, for example inflammatory diseases including psoriasis, and in treating such disease. Host cells and transgenic non-human animals comprising polynucleotides or proteins of the invention are provided. Methods of screening for agents for use in treating corneodesmosin-mediated disease are also provided.

## Background
The present invention relates to nucleotide substitutions, deletions or insertions in the corneodesmosin gene, and the exploitation of these polymorphisms in the detection and/or treatment of corneodesmosin mediated disease, for example inflammatory diseases including psoriasis. The present invention also relates to polynucleotides encoding the corneodesmosin protein, and having one or more nucleotide polymorphisms, and to a protein encoded by said polynucleotides. Also provided are transgenic non-human animals comprising the polynucleotides of the present invention; and methods and kits for treating, diagnosing or determining susceptibility to corneodesmosin mediated disease, in particular by way of gene therapy.
In recent years, it has been recognised that there is considerable genetic diversity in human populations, with common polymorphisms occurring on average at least every kilobase in the genome. Polymorphisms which affect gene expression or activity of the encoded gene product may account for susceptibility to, or expression of, disease conditions, either directly or through interaction with other genetic and environmental factors.
Understanding the molecular basis for disease, by sequencing the human genome and characterising polymorphisms, will enable the identification of those individuals at greatest risk of disease. This will allow the better matching of treatment and disease, and enable the production of new and improved targets for drugs. Screening and treatment of disease may also be better targeted to those in need, thus increasing the cost-effectiveness of health-care provision.
One area in need of such approaches is the diagnosis and treatment of inflammatory diseases. Inflammation, which can be broadly defined as the destructive sequelae to activation of elements of the body's immune system, is a feature of many diseases including infection, autoimmune disorders and benign and malignant hyperplasia. The identification of genetic factors which influence susceptibility to such disorders will provide important new insights into inflammatory disease, and may yield important new diagnostic and/or prognostic tests and treatments.
Psoriasis is a chronic inflammatory cutaneous disorder which affects approximately 2% of the population in the UK and US. Psoriasis manifests itself as red scaly skin patches, principally on the scalp, elbows and knees, and is caused by epidermal hyperproliferation, and abnormal differentiation and infiltration of inflammatory cells. Psoriasis may also be associated with other inflammatory diseases such as arthritis, Crohn's disease, and HIV infection. Population, family, and twin studies all suggest an important genetic component in the pathogenesis of psoriasis, coupled with environmental triggers such as streptococcal infection and stress.
Psoriasis is one of a number of autoimmune diseases that display significant human leukocyte antigen (HLA) associations. The analysis of population-specific HLA haplotypes has provided evidence that susceptibility to psoriasis is linked to the class I and II major histocompatibility complexes (MUC) on human chromosome 6 (Jenisch et al. (1998) Am. J. Hum. Genet 63:191-199). These studies show that psoriasis consists of two distinct disease subtypes (Type I and Type II), which differ in age of onset and in the frequency of HLA types. Type I psoriasis has an age of onset of prior to 40 years and HLA types Cw6, B57, and DR7 are strongly increased. Patients with Type I psoriasis are much more likely to have a positive family history for the disease. In contrast, only about 10% of Cw6-positive individuals develop Type II psoriasis disease, with HLA-Cw2 being over-represented in this group.
Linkage analysis and association studies suggest the presence of a major genetic determinant of psoriasis within the MHC, the strongest candidate gene marker being HLA-C. The most significant association has been shown between HLA-Cw6 and disease Type IA, which has the earliest onset of disease at 0 to 20 years. However, specific involvement of the HLA-Cw6 genotype in disease pathogenesis has yet to be established.
Recently, attention has focussed on non-HLA genes close to HLA-C, in particular the corneodesmosin gene (also known as the S gene), which is located approximately 160 kb telomeric of the HLA-C locus. The corneodesmosin gene consists of 2 exons spanning approximately 5.3 kb of genomic DNA sequence. Two corneodesmosin mRNAs of 2.2 kb and 2.6 kb, resulting from alternative splicing, have been described (Guerrin et al. (1998) J. Biol. Chem. 273:22640-22647). Association studies (Alnini et al. (1999) Hum. Mol. Genet. 8:1135-1140) suggest a strong, significant association between a polymorphism at position 1243 of the corneodesmosin-gene and psoriasis. A corneodesmosin gene haplotype was subsequently defined, which by TDT analysis was shown to have a strong, significant association with psoriasis (Allen et al. (1999) Lancet 353:1589-90).
In human epidermis and other cornified squamous epithelia, corneodesmosin is located in the desmosomes of the upper living layers, and in related structures of the cornified layers, the corneodesmosomes. During maturation of the cornified layers, the protein undergoes a series of cleavages, thought to be a prerequisite of desquamation (shedding of the cuticle or epidermis). Corneodesmosin is detected as a glycosylated and phosphorylated basic protein with an apparent molecular mass of 52-56 kDa. During stratum corneum maturation, corneodesmosin is progressively proteolysed until desquamation occurs. In superficial corneocytes, the 52-56 kDa form is no longer detected and immunoreactive fragments of 45 to 30 kDa predominate; Since location, biochemical characteristics and processing of corneodesmosin are similar in several mammals, it is likely that the protein is essential for the function of corneodesmosomes and corneocyte cohesion. It has been shown that expression of the 56 kDa epidermal keratin polypeptide is increased in psoriatic lesions compared with normal skin and transformation of desmosomes into corneodesmosomes is altered in psoriatic epidermis.
Psoriasis affects approximately 6.4 million people in the US and causes varying ranges of physical discomfort, pain and disability. At present, the causes of psoriasis are unknown. There is no specific test for psoriasis or susceptibility thereto, and diagnosis is based solely on clinical examination and skin histopathology.
It is likely that corneodesmosin is implicated in a range of skin diseases, including psoriasis. In this text, diseases in which corneodesmosin is implicated in the pathology will be referred to as “corneodesmosin-mediated disease”.
The present invention aims to overcome or ameliorate previous limitations in the art by providing means and methods for the detection and treatment of individuals having, or being susceptible to, corneodesmosin mediated disease, in particular inflammatory conditions such as psoriasis.
In a first aspect, the present invention provides an isolated or recombinant polynucleotide comprising a nucleic acid sequence encoding the corneodesmosin gene of FIG. 1, wherein said nucleic acid sequence comprises a nucleotide substitution, deletion or insertion at one or more of positions 6984, 7068, 7077, 7107, 7164, 8884, 8906, 8931, 9538, 9607, 9608, 9647, 9667, 9745, 9761, 9926, 9952, 9968, 10082, 10161, 10162, 10363, 11567, 11641, 11649, 11808, 11839, 11885, 11977, 12018, 12136, 12149, 12198, 12283, 12318, 12345, 12373, 12901, 13001, 13020, 13108, 13117, 13178, 13224, 13316, 13365, 13562, 13605, 13670, 13859, 13889 and 13914 of FIG. 1. (corresponding to positions 284, 368, 377, 407, 464, 2184, 2206, 2231, 2838, 2907, 2908, 2967, 3045, 3061, 3226, 3252, 3268, 3382, 3461, 3462, 3663, 4867, 4941, 4949, 5108, 5139, 5185, 5277, 5318, 5436, 5449, 5498, 5583, 5618, 5645, 5673, 6201, 6301, 6320, 6408, 6417, 6778, 6524, 6616, 6665, 6862, 6905, 6970, 7159, 7189 and 7214 of SEQ ID NO: 1). These novel polymorphisms in the corneodesmosin gene, at the positions indicated above, may be responsible for corneodesmosin mediated disease. In particular, the polymorphisms of the present invention may be useful in identifying individuals susceptible or resistant to corneodesmosin-mediated disease, and in the diagnosis or treatment of such conditions. Preferred combinations of the polymorphisms of the invention are the haplotypes shown in Tables 10a and b. The most preferred haplotype is B of Table 10a.
The polynucleotide of this invention is preferably DNA, or may be RNA or other options.
By “isolated” is meant a polynucleotide sequence which has been purified to a level sufficient to allow allelic discrimination. For example, an isolated sequence will be substantially free of any other DNA or protein product. Such isolated sequences may be obtained by PCR amplification, cloning techniques, or synthesis on a synthesiser. By recombinant is meant polynucleotides which have been recombined by the hand of man.
The corneodesmosin gene sequence shown in FIG. 1 (SEQ ID NO:1) refers to the genomic clone of corneodesmosin, detailed in GenBank Accession No. AC006163 (a genomic clone of the MHC region on chromosome 6p21.3). The single nucleotide polymorphisms of the invention are shown in bold type and underlined on this figure, and have each been given a positional reference with respect to this figure. For reference and comparison with prior art publications, the positional references with respect to the coding sequence have also been given in Table 6, column 2, where nucleotide position 1 corresponds to the first nucleotide of exon 1 and nucleotides upstream of this are given a negative prefix.
A polymorphism is typically defined as two or more alternative sequences, or alleles, of a gene in a population. A polymorphic site is the location in the gene at which divergence in sequence occurs. Examples of the ways in which polymorphisms are manifested include restriction fragment length polymorphisms, variable number of tandem repeats, hypervariable regions, minisatellites, di- or multi-nucleotide repeats, insertion elements and nucleotide deletions, additions or substitutions. The first identified allele is usually referred to as the reference allele, or the wild type. Additional alleles are usually designated alternative or variant alleles. Herein, the sequence exactly as shown in FIG. 1 is designated the reference sequence, and is not part of the invention. Nucleic acid sequences of the present invention which differ from the sequence of FIG. 1 (SEQ ID NO:1) at one or more of the positions indicated above may be referred to as variants of FIG. 1 (SEQ ID NO:1).
A single nucleotide polymorphism is a variation in sequence between alleles at a site occupied by a single nucleotide residue. Single nucleotide polymorphisms (SNP's) arise from the substitution, deletion or insertion of a nucleotide residue at a polymorphic site. Typically, this results in the site of the variant sequence being occupied by any base other than the reference base. For example, where the reference sequence contains a “T” base at a polymorphic site, a variant may contain a “C”, “G” or “A” at that site. Single nucleotide polymorphisms may result in corresponding changes to the amino acid sequence. For example, substitution of a nucleotide residue may change the codon, resulting in an amino acid change. Similarly, the deletion or insertion of three consecutive bases in the nucleic acid sequence may result in the insertion or deletion of an amino acid residue. For ease of reference, where a single nucleotide polymorphism of the present invention results in the insertion or deletion of a nucleotide or amino acid residue, the numbering system of FIGS. 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2) have been maintained.
The single nucleotide polymorphisms of the present invention which occur within the protein coding sequence may contribute to the phenotype of an organism by affecting protein structure or function. The effect may be neutral, beneficial or detrimental, depending upon the circumstances. Whatever the effect, the identification of such polymorphisms enables for the first time determination of susceptibility to disease, and new methods of treatment. The single nucleotide polymorphisms of the invention which occur in the non-coding 5′ or 3′ untranslated regions, may not affect protein sequence, but may exert phenotypic effects by influencing replication, transcription and/or translation. A polymorphism may affect more than one phenotypic trait or may be related to a specific phenotype. In the present invention, polymorphisms in the corneodesmosin gene are likely to affect the phenotype of an individual with respect to corneodesmosin-mediated disease, such as inflammatory disease, in particular psoriasis.
The single nucleotide polymorphisms of the corneodesmosin gene, including those of the present invention, are listed in Table 6 where:

    Column 1 designates each single nucleotide polymorphism a reference number.
    Column 2 provides the positional reference of the polymorphism with respect to FIG. 1.
    Column 3 indicates position of the SNP with respect to the corneodesmosin coding sequence.
    Column 4 shows the location of the polymorphisms in the gene.
    Column 5 shows the sequence flanking the polymorphism, the polymorphism itself being shown in bold type. For example, the polymorphism at position 6984 is shown as C/T, meaning that the variant sequence comprises a T residue, rather than the native C residue.
    Column 6 denotes the standard IUB code.
    Column 7 denotes the SEQ ID NO of the corresponding flanking sequence.


As discussed above, where a single nucleotide polymorphism of the present invention comprises a nucleotide substitution, the substitution may comprise the replacement of the reference base at a polymorphic site with any other base. Each single nucleotide polymorphism described in Table 6, column 4 represents a preferred embodiment of the invention.
It will be appreciated by those skilled in the art that corneodesmosin gene sequences of the invention may comprise one or more nucleotide substitutions, deletions or insertions in addition to one or more of the single nucleotide polymorphisms of the invention.
In a second aspect, fragments of the above polynucleotides are provided, which comprise one or more nucleotide substitutions, insertions or deletions at one or more of the above mentioned positions of FIG. 1 (SEQ ID NO:1). Preferably, a fragment may comprise, or even consist of, the polynucleotide sequence of Table 6, column 4. The novelty of a fragment according to the present embodiment may be easily ascertained by comparing the nucleotide sequence of a fragment with sequences catalogued in databases such as GenBank, or by using computer programs such as DNASIS (Hitachi Engineering, Inc.) or Word Search or FASTA of the Genetic Computer Group (Madison, Wis.).
Preferably, the fragments do not encode a full length protein, as is generally the case with the aforementioned polynucleotides, but otherwise satisfy the requirements of the first aspect. Preferred fragments may be 10 to 150 nucleotides in length. More preferably, the fragments are between 5 to 10, 5 to 20, 10 to 20, 20 to 50, or 50 to 100 nucleotides in length. For example, the fragments may be 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, or 35 nucleotides in length. The fragments may be useful in a variety of diagnostic, prognostic or therapeutic methods, or may be useful as research tools for example in drug screening.
In a third aspect of the invention, there is provided non-coding, complementary sequences which hybridise to the corneodesmosin gene sequence. Such “anti-sense” sequences are useful as probes or primers for-detecting an allele of a polymorphism of the invention, or in the regulation of the corneodesmosin gene. They may also be used as agents for use in the identification and/or treatment of individuals having or being susceptible to corneodesmosin mediated disease.
The anti-sense sequences of the invention include those which hybridise to an allele of a polymorphism of the invention, and also those which hybridise a region flanking the polymorphic site to enable amplification of an allele of one or more polymorphisms. These sequences may be useful as probes or primers. To be useful as a probe, the anti-sense sequence should bind preferentially one allele of one or more polymorphisms of the present invention and will, preferably, comprise the exact complement of one allele of one or more polymorphisms of the invention. Thus, for example, where the variant comprises a “G” residue at position 7068 of FIG. 1 corresponding to posittion 368 of SEQ ID NO:1), it is preferred that the anti-sense sequence will comprise a “C” residue. Such anti-sense sequences which are capable of specific hybridisation to detect a single base mis-match may be designed according to methods known in the art and described in Maniatis et al., Molecular Cloning: A Laboratory Manual 2nd Edition (1989), Cold Spring Harbor, N.Y. and Berger et al., Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) Academic Press Inc. San Diego, Calif.; Gibbs et al., Nuc Acids Res., 17: 2437 (1989); Kwok et al., Nuc Acids Res 18: 999; and Miyada et al., Methods Enzymol. 154: 94 (1987). Variation in the sequence of these anti-sense sequence is acceptable for the purposes of the present invention, provided that the ability of the anti-sense sequence to distinguish between alleles of a polymorphism is not compromised. Similarly, variation in the sequence of a primer sequence is acceptable, provided its ability to mediate amplification of a polymorphic site is not compromised. Preferably, a primer sequence will hybridise to the corneodesmosin gene under stringent conditions which are defined below.
In relation to the present invention, “stringent conditions” refers to the washing conditions used in a hybridisation protocol. In general, the washing conditions should be a combination of temperature and salt concentration so that the denaturation temperature is approximately 5 to 20° C. below the calculated Tm of the nucleic acid under study. The Tm of a nucleic acid probe of 20 bases or less is calculated under standard conditions (1M NaCl) as [4° C.×(G+C)+2° C.×(A+T)], according to Wallace rules for short oligonucleotides. For longer DNA fragments, the nearest neighbour method, which combines solid thermodynamics and experimental data may be used, according to the principles set out in Breslauer et al., PNAS 83: 3746-3750 (1986). The optimum salt and temperature conditions for hybridisation may be readily determined in preliminary experiments in which DNA samples immobilised on filters are hybridised to the probe of interest and then washed under conditions of different stringencies. While the conditions for PCR may differ from the standard conditions, the Tm may be used as a guide for the expected relative stability of the primers. For short primers of approximately 14 nucleotides, low annealing temperatures of around 44° C. to 50° C. are used. The temperature may be higher depending upon the base composition of the primer sequence used.
The anti-sense polynucleotides of this embodiment may be the full length of the corneodesmosin gene of FIG. 1 (SEQ ID NO:1), or more preferably may be 5 to 200 nucleotides in length. Preferred polynucleotides are 5 to 10, 10 to 20, 20 to 50, 50 to 100 or 100 to 200 nucleotides in length. Primers, in particular, are typically 10 to 15 nucleotides long, and may occasionally be 16 to 25.
In a preferred embodiment; the polynucleotides of the aforementioned aspects of the invention may be in the form of a vector, to enable the in vitro or in vivo expression of the polynucleotide sequence. The polynucleotides may be operably linked to one or more regulatory elements including a promoter; regions upstream or downstream of a promoter such as enhancers which regulate the activity of the promoter; an origin of replication; appropriate restriction sites to enable cloning of inserts adjacent to the polynucleotide sequence; markers, for example antibiotic resistance genes; ribosome binding sites: RNA splice sites and transcription termination regions; polymerisation sites; or any other element which may facilitate the cloning and/or expression of the polynucleotide sequence. Where two or more polynucleotides of the invention are introduced into the same vector, each may be controlled by its own regulatory sequences, or all sequences may be controlled by the same regulatory sequences. In the same manner, each sequence may comprise a 3′ polyadenylation site. The vectors may be introduced into microbial, yeast or animal DNA, either chromosomal or mitochondrial, or may exist independently as plasmids. Examples of suitable vectors will be known to persons skilled in the art and include pBluescript II, LambdaZap, and pCMV-Script (Stratagene Cloning Systems, La Jolla (USA))
Appropriate regulatory elements, in particular, promoters will usually depend upon the host cell into which the expression vector is to be inserted. Where microbial host cells are used, promoters such as the lactose promoter system, tryptophan (Trp) promoter system, β-lactamase promoter system or phage lambda promoter system are suitable. Where yeast cells are used, preferred promoters include alcohol dehydrogenase I or glycolytic promoters. In mammalian host cells, preferred promoters are those derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma virus etc. Suitable promoters for use in various host cells would be readily apparent to a person skilled in the art (See, for example, Current Protocols in Molecular Biology Edited by Ausubel et al, published by Wiley).
In a fourth aspect of the present invention there is provided a protein or protein fragment comprising an amino acid substitution, deletion or insertion at one or more of positions 18, 130 or 180 of the amino acid sequence of FIG. 2 (SEQ ID NO:2). Preferably, the protein or protein fragment is encoded by a polynucleotide according to the first aspect of the invention, and comprises a nucleotide insertion, deletion or substitution at one or more of positions 7164, 10082, 10161, 10162 and 10363 of FIG. 1. corresponding to positions 464, 3382, 3461, 3462, and 3663 of (SEQ ID NO:1, respectively). The corneodesmosin protein or protein fragments of the invention may comprise one or more polymorphisms in addition to one or more of the above-mentioned polymorphisms of FIG. 2.
The amino acid sequence exactly as shown in FIG. 2 (SEQ ID NO:2) may be referred to as the reference sequence, and is not part of the invention. The amino acid sequence of FIG. 2 (SEQ ID NO:2) having an amino acid substitution, deletion or insertion at one or more of the positions indicated above may be referred to as a variant of FIG. 2 (SEQ ID NO:2). The reference amino acid at one or more of the above polymorphic sites may be replaced by any other amino acid residue to produce a variant sequence. Amino acid sequences of FIG. 2 (SEQ ID NO:2) having one or more of the polymorphisms disclosed in Table 4 are each preferred embodiments of the invention.
Protein fragments may be functional or non-functional and may be useful in drug screening or gene therapy. Functional fragments may be defined as those which have characteristics of the corneodesmosin protein. The fragments may be at least 10, preferably at least 15, 20, 25, 30, 35, 40 or 50 amino acids in length.
In a fifth aspect of the present invention, there are provided antibodies which react with an antigen of a protein or protein fragment of the fourth aspect. Antibodies can be made by the procedure set forth by standard procedures (Harlow and Lane, “Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1998). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen DNA clone libraries for cells secreting the antigen. Those positive clones can then be sequenced as described in, for example, Kelly et al., Bio/Technology 10:163-167 (1992) and Bebbington et al., Bio/Technology 10:169-175 (1992). Preferably, the antigen being detected and/or used to generate a particular antibody will include proteins or protein fragments according to the fourth aspect.
In a sixth aspect of the present invention, there is provided host cell comprising a polynucleotide according to any of the aforementioned aspects, for expression of the polynucleotide. The host cell may comprise an expression vector, or naked DNA encoding said polynucleotides. A wide variety of suitable host cells are available, both eukaryotic and prokaryotic. Examples include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, preferably immortalised, such as mouse, CHO, HeLa, myeloma or Jurkat cell lines, human and monkey cell lines and derivatives thereof. Such host cells are useful in drug screening systems to identify agents for use in diagnosis or treatment of individuals having, or being susceptible to corneodesmosin mediated disease.
The method by which said polynucleotides are introduced into a host cell will usually depend upon the nature of both the vector/DNA and the target cell, and will include those known to a person skilled in the art. Suitable known methods include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook et al.
In an seventh aspect of the present invention, there is provided a transgenic non-human animal comprising a polynucleotide according to an aforementioned aspect of the invention. Preferably, the transgenic, non-human animal comprises a polynucleotide according to the first or second aspects. The transgenic animal may be either homozygous or heterozygous for the variant sequence. The animal, and cells derived therefrom, are useful for screening biologically active agents that may modulate corneodesmosin function. Such screening methods are of particular use for determining the specificity and action of potential therapies for corneodesmosin mediated disease; such as psoriasis. The animals are useful as a model to investigate the role of corneodesmosin in normal skin function. Transgenic non-human animals are also useful for the analysis of the single nucleotide polymorphisms and their phenotypic effect.
Expression of a polynucleotide of the invention in a transgenic non-human animal is usually achieved by operably linking the polynucleotide to a promoter and/or enhancer sequence, preferably to produce a vector of the fourth aspect, and introducing this into an embryonic stem cell of a host animal by microinjection techniques (Hogan et al., A Laboratory Manual, Cold Spring harbour and Capecchi Science (1989) 244: 1288-1292). Preferably, the construct to be introduced into the animal additionally comprises a) a first homology region with substantial identity to a first corneodesmosin gene sequence; and b) a second homology with substantial identity to a second corneodesmosin gene sequence. The first and second homology regions are of sufficient length for homologous recombination to occur with an endogenous corneodesmosin gene. Those embryonic stem cells comprising the desired polynucleotide sequence may be selected, usually by monitoring expression of a marker gene, and used to generate a non-human transgenic animal. Preferred host animals include mice and other rodents. Further development of such an embryonic stem cell may produce a transgenic animal having cells that are descendant from the embryonic stem cell and thus carry the variant sequence in their genome. Such animals can then be selected and bred to produce animals having the variant sequence in all somatic and germ cells. Such mice can then be bred to homozygosity.
In a preferred embodiment, the transgenic non-human animal may comprise an anti-sense nucleic acid sequence of the third aspect. The expression of an anti-sense sequence in a transgenic non-human animal may be useful in determining the effects of such sequences in treating corneodesmosin-mediated disease, or in neutralising deleterious effects of variant corneodesmosin genes in an animal. Preferably, the host animal will be one which suffers from corneodesmosin mediated disease. The disease may be naturally occurring or artificially introduced.
In some preferred embodiments, for example where the mediated disease has been artificially induced, the transgenic non-human animal will be modulated to no longer expresses the endogenous corneodesmosin gene. Such animals may be referred to as “knock out”. In some cases, it may be appropriate to modulate the expression of the endogenous corneodesmosin gene, or express the polynucleotides of the present invention, in specific tissues. This approach removes viability problems if the expression of a gene is abolished or induced in all tissues. Preferably, the specific tissue would be skin. Where the heterologous gene is human, the animal may be useful in identifying agents which inhibit expression or activity of the variant corneodesmosin sequences of the invention, either in vivo or in vitro.
In an eighth aspect of the present invention there is provided a method of screening for agents for use in the prognosis, diagnosis or treatment of individuals having, or being susceptible to, corneodesmosin-mediated disease, said method comprising contacting a putative agent with a polynucleotide or protein according to an aforementioned aspect of the present invention, and monitoring the reaction there between. Preferably, the method further comprises contacting a putative agent with a reference polynucleotide or protein of FIG. 1 or 2 (SEQ ID NO:1or (SEQ ID NO:2) respectively, and comparing the reaction between (i) the agent and the polynucleotide or protein encoding the reference allele; and (ii) the agent and polynucleotide or protein of the invention. Potential agents are those which react differently with a variant of the invention and a reference allele. It is envisaged that the present method may be carried out by contacting a putative agent with a host cell or transgenic non-human animal comprising a polynucleotide or protein according to the invention. Putative agents will include those known to persons skilled in the art, and include chemical or biological compounds, such as anti-sense polynucleotide sequences, complementary to the coding sequences of the first aspect, or polyclonal or monoclonal antibodies which bind to a product such as a protein or protein fragment of the second aspect. The agents identified in the present method may be useful in determining susceptibility to corneodesmosin-mediated disease, or in the diagnosis, prognosis or treatment of said disease.
In a ninth aspect of the present invention, there is provided a method of diagnosing, or determining susceptibility of a subject to corneodesmosin-mediated disease, said method comprising determining which allele of one or more of the polymorphisms of the invention is present in a subject. The above method may be used in diagnosing or determining susceptibility of a subject to any disease in which corneodesmosin is implicated in the pathology, in particular inflammatory disease, such as psoriasis. The method of the ninth aspect may also be used to identify the presence of a combination of single nucleotide polymorphisms in a subject which define a haplotype linked to corneodesmosin mediated disease. The haplotype may be any particular combination of the above single nucleotide polymorphisms, optionally including known polymorphisms. Preferred haplotypes are those shown in Table 10a, the most preferred haplotype being B of Table 10a.
Any method, including those known to persons skilled in the art, may be used to determine which allele of one or more polymorphisms of the invention is present. Preferably, the method comprises first removing a sample from a subject. More preferably, the method comprises isolating from a sample a polynucleotide or protein to determine therein which allele of one or more polymorphisms of the invention is present.
Any biological sample comprising cells containing nucleic acid or protein is suitable for this purpose. Examples of suitable samples include whole blood, semen, saliva, tears, buccal, skin or hair. For analysis of cDNA, mRNA or protein, the sample must come from a tissue in which the corneodesmosin gene is expressed, and thus it is preferable to use skin samples.
In a preferred embodiment, the method for diagnosing, or determining susceptibility of a subject to a corneodesmosin-mediated disease, comprises determining which allele of one or more polymorphisms of the invention is present, in a polynucleotide. Any method for determining alleles in a polynucleotide may be used, including those known to persons skilled in the art. Preferably, the method may comprise the use of anti-sense polynucleotides, as defined above. Such polynucleotides may include sequences which are able to distinguish between alleles of one or more polymorphisms of the invention, by preferential binding, and sequences which hybridise under stringent conditions to a region either side of a polymorphism of the invention to enable amplification of one or more of the polymorphisms.
Methods of this embodiment include those known to persons skilled in the art, for example direct probing, allele specific hybridisation, PCR methodology including Allele Specific Amplification (ASA), and RFLP.
Determination of an allele of a polymorphism using direct probing involves the use of anti-sense sequences of the third aspect of the invention. These may be prepared synthetically or by nick translation. The anti-sense probes may be suitably labelled using, for example, a radiolabel, enzyme label, fluoro-label, biotin-avidin label for subsequent visualization in, for example, a southern blot procedure. A labelled probe may be reacted with a sample DNA or RNA, and the areas of the DNA or RNA which carry complimentary sequences will hybridise to the probe, and become labelled themselves. The labelled areas may then be visualized, for example by autoradiography.
Allele specific amplification (ASA) discriminates between alleles of a polymorphism on the basis of primers which carry 3′ nucleotides specific for a particular polymorphism. Typically, first and second forward primers are provided, wherein the first forward primer hybridises to one allele of a polymorphism of the invention, and the second forward primer comprises a mis-match at the polymorphic site, thus preventing hybridisation. These primers are used in combination with a backward primer, which hybridises to a distal site to enable amplification of the region between a forward primer and the backward primer. As the first forward primer will only bind to a polymorphic site with which it exhibits perfect complementarity, amplification of the region between the forward and backward primers will indicate the presence of a particular allele. The second forward primer having a is-match at the polymorphic site will not hybridise to the particular allele of a polymorphism, and the absence of a amplification product when this primer is used indicates the absence of the polymorphism. Preferably, the forward primer will be an anti-sense sequence according to the third aspect of the invention. Preferably, the first forward primer will comprise the complement of a single nucleotide polymorphism of the invention at the 3′ most position. The backward primer may hybridise to any suitable portion of the corneodesmosin gene to enable amplification of the intervening region. (see, for example, WO93/22456)
Thus, in a preferred embodiment there is provided a method for diagnosing or determining susceptibility of a subject to corneodesmosin-mediated disease, said method comprising removing a sample from a subject and isolating the nucleic acid therefrom; contacting the sample with either a forward primer which preferentially hybridises to one allele of one or more polymorphisms of the present invention or a forward primer which comprises a mis-match at the polymorphic site and does not hybridise thereto, and a backward primer which hybridises to a distal site; subjecting the nucleic acid sample to amplification; and monitoring for presence of an amplification product which is indicative of the presence of a particular allele of one or more of the polymorphisms of the invention. Preferably, a first reaction is performed using one of the forward primers, and a control reaction is then performed using the other forward primer. It is envisaged that a number alleles of the single nucleotide polymorphisms of the invention may be detected in a single reaction by using multiple primer pairs. Amplification products may then be distinguished by size, using techniques known in the art such as gel electrophoresis, or southern blotting. This method allows the unambiguous identification of individuals homozygous for either allele as well as heterozygous individuals.
“RFLP” refers to restriction fragment length polymorphism and is defined as a method of discriminating between two alleles based upon differences in sequence which result in the presence or absence of a restriction enzyme recognition site. In a preferred embodiment of the present aspect there is provided a method for diagnosing or determining susceptibility to corneodesmosin-mediated disease, said method comprising removing a nucleic acid sample from a subject, and contacting with one or more appropriate restriction enzymes. The size of fragments produced is indicative of which allele of one or more single nucleotide polymorphism according to the invention is present. An allele of a polymorphism of the invention may naturally produce a restriction enzyme site, thus allowing for determination of its presence by analysis of the restriction fragments produced. In some cases, however, an allele of a polymorphism does not create a restriction enzyme site, and one must be artificially introduced. This may be done by using a suitable mis-match primer, according to methods known in the art.
The appropriate restriction enzyme, will, of course, be dependent upon the polymorphism and restriction site, and will include those known to persons skilled in the art. Preferred restriction enzymes are listed in Table 3 (ii), column 11, with the expected fragments sizes in columns 7, 8 and 9. Analysis of the digested fragments may be performed using any method in the art, for example gel analysis, or southern blots.
Preferably, the method may first comprise the amplification of a region of the corneodesmosin gene containing one or more of the polymorphic sites of the invention, for example, using PCR techniques. The probes of the present invention may be useful for this purpose.
The above described methods may require amplification of the DNA sample from the subject, and this can be done by techniques known in the art, such as PCR (see PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY 1992; PCR Protocols: A Guide to methods and Applications (eds. Innis et al., Academic press, San Diego, Calif. 1990); Mattila et al., Nucleic Acids Res. 19-4967 (1991); Eckert et al., PCR Methods and Applications 117 (1991) and U.S. Pat. No. 4,683,202. Other suitable amplification methods include ligase chain reaction (LCR) (Wu et al., Genomics 4 560 (1989); Landegran et al., Science 241 1077 (1988)), transcription amplification (Kwoh et al, Proc Natl Acad Sci USA 86 1173 (1989)), self sustained sequence replication (Guatelli et al., Proc Natl Acad Sci USA 87 1874,(1990)) and nucleic acid based sequence amplification (NASBA). The latter two methods both involve isothermal reactions based on isothermal transcription which produce both single stranded RNA and double stranded DNA as the amplification products, in a ratio of 30 or 100 to 1, respectively.
It may often be desirable to identify the presence of multiple single nucleotide polymorphisms in a sample from a subject. This may be the case in the present invention where the corneodesmosin gene contains 39 polymorphisms, each of which may be indicative of a different phenotype. For this purpose, nucleic acid arrays may be useful, as described in WO95/11995. The array may contain a number of probes, each designed to identify one or more of the above single nucleotide polymorphisms of the corneodesmosin gene, as described in WO95/11995.
In a further preferred embodiment of the ninth aspect, the method may comprise determining which allele of one or more polymorphisms is present in a protein of the invention Any method for determining the presence of a particular form, or allele, of a protein is present, may be used. One such method involves the use of antibodies in diagnosing or determining susceptibility to corneodesmosin mediated disease. The method may comprise removing a sample from a subject, contacting the sample with an antibody to an antigen of a protein or protein fragments according to the second aspect of the present invention, and detecting binding of the antibody to the antigen, wherein binding is indicative of the presence of a particular allele or form of the protein and thus risk to corneodesmosin mediated disease. Tissue samples as described above are suitable for this method.
The detection of binding of the antibody to the antigen in a sample may be assisted by methods known in the art, such as the use of a secondary antibody which binds to the first antibody, or a ligand. Immunoassays including immunofluorescence assays (IFA) and enzyme linked immunosorbent assays (ELISA) and immunoblotting may be used to detect the presence of the antigen. For example, where ELISA is used, the method may comprise binding the antibody to a substrate, contacting the bound antibody with the sample containing the antigen, contacting the above with a second antibody bound to a detectable moiety (typically an enzyme such as horse radish peroxidase or alkaline phosphatase), contacting the above with a substrate for the enzyme, and finally observing the colour change which is indicative of the presence of the antigen in the sample.
In a tenth aspect of the invention, there is provided a method of treating a subject who has been diagnosed as having, or being susceptible to, corneodesmosin mediated disease such as psoriasis. The mode of treatment will depend upon the nature of the polymorphism(s) and the phenotypic effect, and preferably comprises negating the effect of the disease causing polymorphism(s). Where a subject has been diagnosed according to the methods of the invention, treatment to negate the effect of the disease causing polymorphism may include any suitable means. A suitable treatment includes the administration of a polynucleotide sequence which hybridises, preferably under stringent conditions (as defined above), to the corneodesmosin gene. Such polynucleotide sequences may include the anti-sense sequences of the third aspect. Alternatively, the treatment may comprise a polynucleotide sequence encoding the corneodesmosin gene or a fragment thereof, and having either a reference or variant allele of a polymorphism of the invention. Preferably, the method, comprises:
(i) determining which allele of one or more polymorphisms of the invention are present; and
(ii) administering a polynucleotide sequence which hybridises under stringent conditions to the corneodesmosin gene; or a polynucleotide sequence encoding the reference sequence of the corneodesmosin gene or a fragment thereof, or a polynucleotide sequence of the first aspect.
In an alternative embodiment of this aspect, there is provided the use of a polynucleotide sequence of the tenth aspect in the manufacture of a medicament for use in the diagnosis and treatment of corneodesmosin mediated disease.
This method of diagnosis and treatment may comprise determining and introducing alleles in the form of a polynucleotide or protein. In the above embodiments, the allele of a polymorphism may be determined using any method, as discussed above. The treatment may be introduced in the form of a protein, or polynucleotide. Any suitable means for introduction of a protein may be used. Introduction of a polynucleotide may use gene therapy methods including those known in the art. In general, a polynucleotide encoding the allele will be introduced into the target cells of a subject, usually in the form of a vector and preferably in the form of a pharmaceutically acceptable carrier. Any suitable delivery vehicle may be used, including viral vectors, such as retroviral vector systems which can package a recombinant genome. The retrovirus could then be used to infect and deliver the polynucleotide to the target cells. Other delivery techniques are also widely available, including the use of adenoviral vectors, adeno-associated vectors, lentiviral vectors, pseudotyped retroviral vectors and pox or vaccinia virus vectors. Liposomes may also be used, including commercially available liposome preparations such as Lipofectin®, Lipofectamine®, (GIBCO-BRL, Inc. Gaitherburg, Md.), Superfect® ((Qiagen Inc, Hilden, Germany) and Transfectam® (Promega Biotec Inc, Madison Wis.).
The polynucleotide or vehicle may be administered parenterally (eg, intravenously), transdermally, by intramuscular injection, topically or the like. As corneodesmosin mediated diseases are usually manifested in the skin, topical administration is preferred. The exact amount of polynucleotide or vehicle to be administered will vary from subject to subject and will depend upon age, weight, general condition, and severity or mechanism of the disorder.
In a further aspect, the present invention provides a kit for the detection in a subject of a single nucleotide polymorphism according to the present invention. Preferably, the kit will contain polynucleotides according to the aforementioned aspects, most preferably the anti-sense sequences of the third aspect for use as probes or primers; antibodies of the fifth aspect; or restriction enzymes for use in detecting the presence of a polynucleotide, protein or protein fragment of the invention. Preferably, the kit will also comprise means for detection of a reaction, such as nucleotide label detection means, labelled secondary antibodies or size detection means. In yet a flirter preferred embodiment, the polynucleotides, or antibodies may be fixed to a substrate, for example an array, as described in WO95/11995.
The preferred embodiments of each aspect apply to the other aspects of the invention, mutatis mutandis.



The present invention will now be described by way of a non-limiting example, with reference to the following figures in which:
FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) of the genomic clone of the corneodesmosin gene, of GenBank Accession No. AC006163.
FIG. 2 shows the amino acid sequence (SEQ ID NO:2) of the corneodesmosin protein and coding sequence therefor.
FIG. 3 shows the exon and intron structure of the corneodesmosin gene.



EXAMPLES
Determination of Gene Structure
The mRNA sequence of the corneodesmosin gene (GenBank Accession ID NM—001264) was used to screen the following public DNA databases: (available through the National Centre for Biotechnology Information website—http://www.ncbi.nlm.nih.gov/); NR (Non-Redundant DNA), HTGS (High Throughput Genomic Sequence), and GSS (Genome Survey Sequence). The analysis was performed using the BLASTN algorithm (Altschul, et al., (1990) J. Mol. Biol. 215-403-410). Any genomic sequences containing the corneodesmosin gene were identified by their degree of sequence identity. The gene structure was determined by comparison of the mRNA sequence with the genomic clones. The deduced exon-intron organisation of the corneodesmosin gene is presented in FIG. 3.
Oligonucleotide Primer Design for Corneodesmosin Gene Sequencing
5 pairs of oligonucleotide primers (S1F/S1R; S2.1F/S2.1R; S2.2F/S2.2R; S2.3F/S2.3R; S2.4F/S2.4R, S2.5F/S2.5R Table 1) were designed to amplify exons 1 and 2 of the corneodesmosin gene including 350 bp 5′ untranslated region (UTR) and 909 bp 3′ UTR sequences. Oligonucleotide primer sequences were derived from human chromosome 6p21 genomic DNA sequence (GenBank Accession AC006163).







TABLE 1







Oligonucleotide Primer DNA Sequences.









Primer ID
Primer Sequence
SEQ ID NO












S 1F  
dCTGGGTCCCGTGGCAAGA
5


 


S 1R  
dGTCCTCTCCCGGAGTCTC
6


 


S 2.1F
dGGTGAGGGAGGAAGCCAAG
7


 


S 2.1R
dGAGCTGACGCTTTGGCCAC
8


 


S 2.2F
dGCCAACCAATGACAACTCTTACC
9


 


S 2.2R
dGCCTCCACAGAGCTGGAC
10


 


S 2.3F
dGGCAAATACTTCTCCAGCAACC
11


 


S 2.3R
dGGCCTTCTCCCATATGGGA
12


 


S 2.4F
dCCAAGGAGAGTTACTCGACAG
13


 


S 2.4R
dGGCATATTGGGTGGGTTGAC
14


 


S 2.5F
dCATCTGGAAACAGTGGCCAC
15


 


S 2.5R
dGTCTTCCTCCTCTGTGGGAG
16









Corneodesmosin Gene Amplification

Genomic DNA from a panel of 24 unrelated individuals was amplified using primer pairs S1F/S1R; S2.1F/S2.1R; S2.2F/S2.2R; S2.3F/S2.3R; S2.4F/S2.4R, S2.5F/S2.5 R. 100 ng genomic DNA was amplified by PCR in a total reaction volume of 25 μl containing 50 mM KCl, 20 mM Tris.HCl (pH 8.4), 2 mM MgCl2 200μM each dATP, dCTP, dGTP, dTTP, 1 μM each oligonucleotide primer and 0.5 units AmpliTaq Gold DNA polymerase (Applied Biosystems). Reactions were thermocycled with an initial denaturation step of 95° C./10 mins followed by 35 cycles of 94° C./30 secs; Tm annealing/30 secs; 72° C./30 secs. A final elongation step of 72° C./10 mins completed the amplification. Annealing temperatures (Tm) for specific primer pairs are presented in Table 2.







TABLE 2







Primer Annealing Temperatures and Amplimer Sizes.










Amplimer
Primer Pairs
Fragment size (bp)
Tm (° C.)













1
S1F and S1R
495
63


2.1
S2.1F and S2.1R
610
62


2.2
S2.2F and S2.2R
619
62


2.3
S2.3F and S2.3R
621
63


2.4
S2.4F and S2.4R
532
59


2.5
S2.5F and S2.5R
474
61









Heteroduplex Analysis using DHPLC:

Oligos were designed to amplify products of between 400-800 bp in length from the genomic DNA of 12-25 individuals. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed using the WAVETM DNA fragment analysis system (Transgenomic) (Kuklin, et al., (1997-98) Genet Test. 1(3):, 201-6.). The temperature required for successful resolution of heteroduplex molecules within each PCR product was determined empirically by injecting PCR product at a series of increasing mobile phase temperatures and constructing a fragment specific melting curve. A universal gradient for double stranded DNA was used to determine the appropriate acetonitrile concentration for the heteroduplex identification. For mutation detection, 1-2 μl aliquots of the PCR reactions from each of the eleven individuals were injected onto the WAVETM column. Mutation detection gradients were for four minutes. Results were graphically visualised using the D-7000 HSM software (Transgenomic).
Direct Sequencing of PCR Products
50-100 ng of PCR products were sequenced in both orientations using the DYEnamic ET terminator cycle sequencing premix kit from Amersham. Reactions were fractionated on ABI 377 automated sequencers using standard procedures. Chromatographic traces were analysed using the SEQUENCHER programme (Gene Codes, USA), to identify SNP positions.
Detection of Variant Alleles—Assay Design for Genotyping
The fragment sequence containing the polymorphism was analysed for the creation or deletion of a naturally occurring restriction enzyme recognition site in response to variation in the nucleotide sequence. If the polymorphism did not result in any changes in restriction enzyme recognition sites then the sequence was interrogated with the Primer Design Mismatch ProgramTM. This is an adaptation of the program described by Davidow L S ((1992) Comput Appl Biosci 8:193-194).
Detection of Polymorphisms in 24 Population Controls
The application of the approach outlined above resulted in the identification of 39 SNPs. These are described in Table 3, in which:

    Column 1 designates each single nucleotide polymorphism a reference number.
    Column 2 provides the positional reference of the polymorphism with respect to FIG. 1, together with details of the polymorphism itself. For example, the reference “C6948T” indicates a substitution of the nucleotide “C” for nucleotide “T” at position 6984 of FIG. 1.
    Column 3 of (i) provides the corresponding positional references with respect to the coding sequence of the corneodesmosin gene.
    Column 4 of (i) indicates the region of the gene which the polymorphism occurs.
    Column 5 of (i) shows the sequence flanking the polymorphism, the polymorphism itself being shown in bold type. The single nucleotide polymorphisms are defined using standard IUB code.
    Column 6 of (i) indicates the SEQ ID NO of the corresponding flanking sequence.
    Columns 3 and 5 of (ii) show primer sequences which may be used to amplify a region of the corneodesmosin gene to enable detection of the single nucleotide polymorphism by using restriction enzyme analysis. The amplified product size is shown in Column 7 of (ii).
    Columns 4 to 6 of (ii) indicate the SEQ ID NO of the corresponding primer sequence.
    Columns 8 to 10 of (ii) list the restriction enzymes used to digest the amplified product, and the sizes of fragments generated by the reference, variant and heterozygous sequences respectively.


RFLP or ASA assays were developed for all of these SNPs and the corresponding primers along with amplification product and digestion fragment sizes are also given in Table 3. Of these 39 SNPs, 9 give rise to amino acid changes. These are shown in Table 4.
Additional Corneodesmosin Polymorphisms
In a subsequent experiment, DNAs from 96 individuals comprising 24 type TA psoriatics, 24 type MB psoriatics, 24 type II psoriatics and an additional 24 healthy controls, were sequenced as described above using primers designed to cover the remainder of the Corneodesmosin gene (see Table 5a)
The sequencing reactions were carried out with 50-100 ng of PCR products sequenced in both orientations using the DYEnamic ET terminator cycle sequencing premix kit from Amersham according to the following protocol:
The PCR products were Exo/Sap treated and desalted using p10 columns, prior to setting up the sequencing reactions in a thermowell plate including:

    
    
        200-400 ng PCR Product
        1 μl primer@ 10 pmolml−1 
        8 μl ET Termination mix
        H2O to 20 μl
    
    


The plates were sealed with an MJ Research Microseal film and then vortexed to mix samples, followed by a spin to ensure reaction is at the bottom of the wells.
PCR was carried out according to the following protocol:

    
    
        No Predenaturation
        95° C. for 30 sec
        50° C. for 15 sec
        60° C. for 1 min
        for 40 cycles and then hold at 10° C. until ready to purify.
    
    


After removing the plate from the thermocycler, the products were purified by ethanol precipitation. To each well we added 2 μl 7.5M ammonium acetate followed by 80 μl 100% ethanol and incubated at room temperature for 10 minutes before spinning at 4000 rpm for 1 hour at room temperature. The supernatant was discarded and the pellet washed with 70% ethanol before centrifugation for a further 30 minutes. The supernatant was discarded and remaining ethanol removed gently by pipetting using p10 tips before allowing the pellets to air dry.
The samples were then resuspended in 10 μl MegaBACE Loading Buffer (Molecular Dynamics) and transferred to a Robbins plate prior to loading onto the MegaBACE. Reactions were fractionated on a Molecular Dynamics MegaBACE capillary sequencer using standard procedures. Chromatographic traces were analysed using the SEQUENCHER programme (Gene Codes, USA), to identify SNP positions.
A total of 28 novel SNPs were identified (additional to those given in the example above). For reference, these are SNPs 6-18 and 53-67 in Table 5b. A combined list of Corneodesmosin SNPs is given in Table 6.
Corneodesmosin Gene Association with Psoriasis
A total of 21 SNPs (see Table 7) were genotyped in 147 families identified through a proband with psoriasis (a total of 499 individuals, of whom 233 were affected). The genotyping was carried out using a variety of methods (single base extension using the Snapshot kit from Amersham Pharmacia Biotech, Pyrosequencing (Ahmadian A et al., Anal Biochem 2000 280:103-10), or direct sequencing) as given in Table 7. All these methods used established methodologies that are provided by the equipment manufacturers and/or are well known to those skilled in the art.
Linkage Disequilibrium
The extent of linkage disequilibrium (LD) between markers was calculated using genotype data from 199 unrelated, unaffected individuals and is expressed as correlation coefficients in Table 8. This analysis shows that there is extensive linkage disequilibrium between many of the Corneodesmosin polymorphisms.
Single Point Association
Single point associations between each SNP and psoriasis affected status were calculated using the TRANSMIT program (Clayton D, MRC Biostatistics Unit, Cambridge)—see Table 9. Highly significant associations were observed between SNPs 19, 21, 23, 24, 26, 28, 30, 33, 34, 37, 38 and psoriasis. The single SNP showing the most significant association with psoriasis that has been previously reported is SNP 33 (Tazi Ahnini R et al, Hum. Mol. Genet. 1999: 8 pp 1135-40; Allen M H et al, Lancet 1999: 353 pp1589-90).
This study has identified 9 SNPs, (19, 21, 24, 26, 28, 30, 34, 37 and 38) which show global chi-squared values greater than that seen for SNP 33, and are therefore more powerfully predictive of affected status.
Haplotype Analysis
A total of 19 SNPs were used for haplotype analysis (SNPs at positions 29 and 32 were excluded due to low information content). Three common haplotypes were identified.(Table 10). Of the three common haplotypes, haplotype B is significantly associated with psoriasis. The alleles are coded alphabetically (Table 10b) such that the nucleotide first in the alphabet is given coded as 1, and the other nucleotide is coded as 2. Thus A is always 1, T is always 2, and G or C are coded depending on the other nucleotide. For example, in SNP No. 1, which is a C to T substitution, the presence of the C allele is coded as 1 and the presence of the T allele is coded as 2 (see Table 10b). In Table 10a, this means that haplotypes A and B have C residues, and haplotype C has a T residue at this position. For an A to C substitution, the A allele will be coded as 1, and the C allele as 2. In a C to G substitution, the C allele will be 1 and the G allele 2.
Construction of Corneodesmosin Gene Targeting Vector
As the genetic data pointed strongly to an involvement of the Corneodesmosin gene in the pathophysiology of psoriasis, we decided to engineer mouse strains in which the mouse orthologue of the corneodesmosin gene is knocked out by homologous recombination using a vector construct designed to remove exon 2 of the Corneodesmosin gene.
Murine Corneodesmosin genomic clones were isolated from a mouse large insert PAC library, using mouse Corneodesmosin cDNA sequence as a probe by standard techniques. The isolated murine Corneodesmosin genomic clones were then restriction mapped in the region of the Corneodesmosin gene using small oligonucleotide probes and standard techniques. The murine genomic locus was partially sequenced to enable the design of homologous arms to clone into the targeting vector. The murine Corneodesmosin gene is a two-exon gene. A 4 kb 5′ homologous arm and a 1 kb 3′ homologous arm where amplified by PCR and the fragment cloned into the targeting vector. The position of these arms was chosen to functionally disrupt the Corneodesmosin gene by deleting the majority of the coding sequence. A targeting vector was prepared where the deleted Corneodesmosin sequence was replaced with non-homologous sequences composed of an endogenous gene expression reporter (an in frame fusion with lacZ) upstream of a selection cassette composed of a self promoted neomycin phosphotransferase (neo) gene in the same orientation as the Corneodesmosin gene.
Transfection and Analysis of Embryonal Stem Cells
Embryonal stem cells (Evans M J & Kaufman M H Nature 1981 292:154-6) were cultured on a neomycin resistant embryonal fibroblast feeder layer grown in Dulbecco's Modified Eagles medium supplemented with 20% Fetal Calf Serum, 10% new-born calf serum, 2 mM glutamine, non-essential amino acids, 100 μM 2-mercaptoethanol and 500 u/ml leukemia inhibitory factor. Medium was changed daily and ES cells were subcultured every three days. 5.times.10.sup.6 ES cells were transfected with 5 μg of linearized plasmid by electroporation (25 μF capacitance and 400 Volts). 24 hours following electroporation the transfected cells were cultured for 9 days in medium containing 200 μg/ml neomycin. Clones were picked into 96 well plates, replicated and expanded before being screened by PCR to identify clones in which homologous recombination had occurred between the endogenous Corneodesmosin gene and the targeting construct. From 96 picked clones 45 targets were identified. These clones where expanded to allow replicas to be frozen and sufficient high quality DNA to be prepared for Southern blot confirmation of the targeting event using external 5′ and 3′ probes, all using standard procedures (Russ et al. Nature 404:95-99).
Generation of Corneodesmosin Deficient Mice
C57BL/6 female and male mice were mated and blastocysts were isolated at 3.5 days of gestation. 10-12 cells from Clone 7 (described in Example 2) were injected per blastocyst and 7-8 blastocysts were implanted in the uterus of a pseudopregnant F1 female. Five chimeric pups were born of which one male was 100% agouti (indicating cells descendent from the targeted clone). This male chimera was mated with female and MF1 and 129 mice, and germline transmission was determined by the agouti coat color and by PCR genotyping respectively.
Corneodesmosin Knock-out Mouse as a Model of Corneodesmosin-mediated Disease
Mice heterozygous for the Corneodesmosin knockout are superficially normal. Staining for expression of the lacZ reporter gene fused to the Corneodesmosin promoter in the knockout construct shows clear expression in desquamating skin. We then genotyped surviving offspring from intercrosses of heterozygous knockout mice on an outbred genetic background in an attempt to isolate mice homozygous for the knockout.
From 44 surviving progeny we identified:

    
    
        17 wild type
        27 heterozygotes
        0 homozygous mutant.
    
    


Statistical analysis of these data indicate that the ratio of wild type:heterozygous animals conforms to a 1:2 ratio consistent with a homozygous lethal phenotype (Chi square 0.557).
In keeping with this analysis, two pups found dead 24-48 hours after birth were homozygous mutant. Together these data indicate the Corneodesmosin deficiency in mice is lethal with pups dying soon after birth, most likely through dehydration as a result of failure to establish a permeability barrier in the skin.
We conclude from this that altering the activity of Corneodesmosin (e.g. by modulating expression or altering its proteolytic processing) will be useful in developing models of disease in which epithelial integrity is increased (e.g. psoriasis) or decreased (e.g. dermatitis), and for testing novel agents for the alleviation of Corneodesmosin mediated disease.







TABLE 3







(i) S Gene SNPs with location and assay details













SNP
Corneodesmosin


SEQ


SNP
nt position
nt position
Location
Flanking Sequence
ID NO





1
C6984T
−115
5′UTR
CTCCCGGCCA CACCAACTTC CCCCYGGGCA CCCACCCCCT CCACCTCTCC
17


 


2
A7068G
−31
5′UTR
AATGTCCAGCTCTGGCATAA AGGACCCRGG TGTCCTCGAG CTGCCATCAG
18


 


3
C7077T
−22
5′UTR
TCTGGCATAA AGGACCCAGG TGTCCTYGAG CTGCCATCAG TCAGGAGGCC
19


 


4
C7107T
9
5′UTR
CTGCCATCAG TCAGGAGGCCGTGCAGYCCG AGATGGGCTC GTCTCGGGCA
20


 


5
A7164T
86
Coding
GGCGTGTGGGTGGGCACGGG ATGWTGGCAC TGCTGCTGGC TGGTCTCCTC
21





Sequence


 


6
C10039T
137
Coding
CTAAGAGCAT TGGCACCTTC TCAGACCCYT GTAAGGACCCCACGCGTATC
22





Sequence


 


7
C10082T
180
Coding
ACCTCCCCTAACGACCCCTGCYTCACTGGGAAGGGTG
23





Sequence


 


8
C10134T
206
Coding
CAGTAGCTAC AGTGGCTCCA GCAYTTCTGG CAGCTCCATTTCCAGTGCCA
24





Sequence


 


9
G10344A
442
Coding
GAGCAGCAGC TCTCACTCGG GAARCAGCGGCTCTCACTCG GGAAGCAGCA
25





Sequence


 


10
10363(AAG)ins
461
Coding
GAAGCAGCGGCTCTCACTCG GG(AAG)CAGCA GCTCTCATTCGAGCAGCAGC
26





Sequence


 


11
A10516G
614
Coding
CTGGACAAAGCTCTTCCTCT TCCCARACCT CTGGGGTATC CAGCAGTGGC
27





Sequence


 


12
C10521T
619
Coding
CTGGACAAAGCTCTTCCTCT TCCCAAACCT YTGGGGTATC CAGCAGTGGC
28





Sequence


 


13
T10624C
722
Coding
GGAGGGCCCA TCGTCTCGCA CTCYGGCCCC TACATCCCCA GCTCCCACTC
29





Sequence


 


14
G10669A
767
Coding
GCTCCCACTCTGTGTCAGGG GGTCAGAGRC CTGTGGTGGT GGTGGTGGAC
30





Sequence


 


15
T10873C
971
Coding
CCTACAGTAA GGGTAAAATC TAYCCTGTGG GCTACTTCAC CAAAGAGAAC
31





Sequence


 


18
G11020A
1118
Coding
AGCCAGTCGGCAGCTTCCTC GGCCATTGCR TTCCAGCCAG TGGGGACTGG
32





Sequence


 


17
A11117G
1215
Coding
CTCCCTCCAGTTCTCGAGTC CCCAGCRGTT CTAGCATTTC CAGCAGCTCC
33





Sequence


 


18
T11138G
1236
Coding
CCCAGCAGTTCTAGCATTTC CAGCAGCKCC GGTTCACCCTACCATCCCTG
34





Sequence


 


19
G11142T
1240
Coding
CTAGCATTTC CAGCAGCTCC GKTTCACCCTACCATCCCTGCGGCAGTGCT
35





Sequence


 


20
C11145T
1243
Coding
CTAGCATTTC CAGCAGCTCC GGTTYACCCTACCATCCCTGCGGCAGTGCT
36





Sequence


 


21
G11233C
1331
Coding
GCAGCAGCTC CAGTTCCCAA TCSAGTGGCA AAATCATCCTTCAGCCTTGT
37





Sequence


 


22
T11260C
1358
Coding
TCGAGTGGCA AAATCATCCTTCAGCCTTGY GGCAGCAAGT CCAGCTCTTC
38





Sequence


 


23
G11495A
1593
Coding
TTCCTACCCC AAGGAGAGTT ACTCRACAGTCCATAAGTCA ACTGTTGTGT
39





Sequence


 


24
11505(AAG)ins
1603
3′UTR
GAGAGTTACTCGACAGTCCATAAG(AAG)TCAACTGTTGTGTGTGTGCATGC
40


 


25
G11576T
1674
3′UTR
TACACTATATCCCATATGGGAGAAGKCCAGTGCCCAGGCATAGGGTTAGC
41


 


26
T11641C
1739
3′UTR
CCCAAAAGAGTGGTTCTGCTTTCTCYACTACCCTAAGGTTGCAGACTCTC
42


 


27
T11649C
1747
3′UTR
AGTGGTTCTGCTTTCTCTACTACCCYAAGGTTGCAGACTCTCTCTTATCA
43


 


28
T11808G
1906
3′UTR
CCCCTTACAATTCCCTCTACTGTGTKGAAATGGTCCATTGAGTAACACCC
44


 


29
C11839G
1937
3′UTR
GGTCCATTGAGTAACACCCCCATCASCTTCTCAACTGGGAAACCCCTGAA
45


 


30
C11885T
1983
3′UTR
TGAAATGCTCTCAGAGCACCTCTGAYGCCTGAAGAAGTTATACCTTCCTC
46


 


31
C11977T
2075
3′UTR
AAACAGTGGC CACTTTTCAC TGACCTYTCT TCGACATCTA GTCAACCCAC
47


 


32
T12018C
2116
3′UTR
CAACCCACCCAATATGCCACTGGGCYTTCGCTCCCAATTCCACCCCACCC
48


 


33
T12136C
2234
3′UTR
TTATCTCAGCCCCTTCCTGTGGCCAYTTCCCTCAGTGCCCAGATGATTCC
49


 


34
C12149T
2247
3′UTR
TTCCTGTGGCCATTTCCCTCAGTGCYCAGATGATTCCCTGGGTGAGGGAG
50


 


35
G12198A
2296
3′UTR
GACACTGGGGCACCCTCAGAGGTTGRAGCAGGCTCCCTGCTGTCCCTGGA
51


 


36
G12283A
2381
3′UTR
GGTGCAGACTTTTTGCCTTCTTGGARTCCTGGGTCTCCTCTGAGAGTCTG
52


 


37
T12318C
2416
3′UTR
TCCTCTGAGAGTCTGGGTGGTGCTCYTCCTACGCCTCTAGAGGTCTCTGT
53


 


38
C12345T
2443
3′UTR
CCTACGCCTCTAGAGGTCTCTGTGTYCCTCATTTTCCTTCAAAAGCGGGC
54


 


39
G12373A
2471
3′UTR
TCATTTTCCTTCAAAAGCGGGCTGTRTTTCTCTTCTACCTTCCAGCTCCT
55










(ii)
















SEQ

SEQ




SNP

ID

ID
PCR product


SNP
nt position
Primer sequence
NO
Primer sequence
NO
size (bp)





1
C6984T
dCTGGGTCCCGTGGCAAGA
5
dGTCCTCTCCCGGAGTCTC
6
495


 


2
A7068G
dCTGGGTCCCGTGGCAAGA
5
dCTGACTGATGGCAGCTCGAGGACAGC
58
333


 


3
C7077T
dCTGGGTCCCGTGGCAAGA
5
dGTCCTCTCCCGGAGTCTC
6
496


 


4
C7107T
dCCCACCCCCTCCACCTCT
59
dCCGTCCCCTTCGCTGGGTCCTC
60
283


 


5
A7164T
dATTACCACGCTCCTCCCG
61
dGCAGGAGGAGACCAGCCAGCAGCAGTGTCA
62
249


 


6
C10039T
dCAGTTCTTCCTCCTTTCTCCAT
63
dAGGGGAGGTGATACGCGTGGGGTCCTTCCA
64
215


 


7
C10082T
dGACCTTGGCTAAGAGCATTG
65
dCCTGGCTTAAAAGATCCTGC
66
240


 


8
C10134T
dGGTGAGGGAGGAAGCCAAG
7
dAGAACTGCTGGAGCCACTGTAGCTACTGCA
68
193


 


9
G10344A
dCAGCTGGGGAGCAGCAGCTCTCCCTCGGGA
69
dGAGCTGACGCTTTGGCCAC
8
243


 


10
10363(AAG)ins
dAGCGGCTCTCACTCGGGAAG
71
dTGACGCTTTGGCCACTGCTG
72
204


 




dAGCGGCTCTCACTCGGGCAG
73


 


11
A10516G
dCAGCCTGGACAAAGCTCTTCCTCTTCTCA
74
dCTGGAAGGCCACCATTGCTA
75
269


 


12
C10521T
dGCCAACCAATGACAACTCTTACC
9
dGCCTCCACAGAGCTGGAC
10
620


 


13
T10624C
dCTGCAGTGGAGGGCCCATCGTCTCGCACAC
78
dCTGGAAGGCCACCATTGCTA
79
162


 


14
G10669A
dGCCAACCAATGACAACTCTTACC
76
dGCCTCCACAGAGCTGGAC
77
620


 


15
T10873C
dAGGCATGACCTACAGTAAGGGTAAAATCGA
80
dGCCTCCACAGAGCTGGAC
77
221


 


16
G11020A
dCAGCCAGTCGGCAGCTTCCTCGGCCATCGC
81
dTGAAGGAGCCGGTGCCTG
82
225


 


17
A11117G
dTGCTCTCCCTCCAGTTCTCGAGTCCCCTGC
83
dGTGTCAAGGAGGAGACAGACA
84
231


 


18
T11138G
dGGCAAATACTTCTCCAGCAACC
11
dGGCCTTCTCCCATATGGGA
12
622


 


19
G11142T
dGGCAAATACTTCTCCAGCAACC
11
dGGCCTTCTCCCATATGGGA
12
622


 


20
C11145T
dGTTCTAGCATTTCCAGCAGCTCCGATT
87
dGTGTCAAGGAGGAGACAGACA
88
200


 


21
G11233C
dGGCAAATACTTCTCCAGCAACC
11
dGGCCTTCTCCCATATGGGA
12
622


 


22
T11260C
dGGCAAATACTTCTCCAGCAACC
11
dGTGACCAGAAGAGCTGGACTTGCTGGC
89
331


 


23
G11495A
dGGCAAATACTTCTCCAGCAACC
11
dGGCCTTCTCCCATATGGGA
12
622


 


24
11505(AAG)ins
dGGAGAGTTACTCGACAGTCCATAAGAAG
90
dCAGTAGGAGAGAATCAAGAGAGGAGC
91
259


 




dGGAGAGTTACTCGACAGTCCATAAGTCA
92
dCAGTAGGAGAGAATCAAGAGAGGAGC
91


 


25
G11576T
dAAGGAGAGTTACTCGACAGTCC
93
dAGGAGAGAATCAAGAGAGGAGC
94
254


 


26
T11641C
dAAGGAGAGTTACTCGACAGTCC
93
dTAAGAGAGAGTCTGCAACCTTAGGGTAGC
95
190


 


27
T11649C
dAAGGAGAGTTACTCGACAGTCC
93
dAGGAGAGAATCAAGAGAGGAGC
94
254


 


28
T11808G
dAGGTTGCAGACTCTCTCTTATCACCC
96
dATGGGGGTGTTACTCAATGGACCATGTC
97
186


 


29
C11839G
dAGGTTGCAGACTCTCTCTTATCACCC
96
dAAGTGGCCACTGTTTCCAGATGATGG
98
315


 


30
C11885T
dAGGTTGCAGACTCTCTCTTATCACCC
96
dAAGTGGCCACTGTTTCCAGATGATGG
98
315


 


31
C11977T
dCCATCATCTGGAAACAGTGG
99
dCGTGGTGAGCTCTGTAATGG
100
124


 


32
T12018c
dACCATCATCTGGAAACAGTGGC
101
dTGAGCTCTGTAATGGAGGGTGG
102
120


 


33
T12136C
dCCTTATCTCAGCCCCTTCCTGTGGCCT
103
dATCTGTCCAGGATCCAGGGACAGC
104
126


 


34
C12149T
dAACACACCCATTGCCTCTCAAG
105
dCCACAGTTTACTGAGCCATCTG
106
167


 


35
G12198A
dCCTTATCTCAGCCCCTTCCTGTGGC
107
dAGGATCCAGGGACAGCAGGGAGCCTGGT
108
118


 


36
G12283A
dGGACAGATGGCTCAGTAAACTG
109
dAGGGACACAGAGACCTCTAG
110
122


 


37
T12318C
dGGACAGATGGCTCAGTAAACTG
109
dAGGGACACAGAGACCTCTAG
110
122


 


38
C12345T
dCTCTTCCTACGCCTCTAGAGGTCTCTGGGT
111
dGCAATGAGAGAGGAGGGAAATGGCG
112
179


 


39
G12373A
dGTCCCTCATTTTCCTTCAAAAGCGGGCAG
113
dGGGAAGAGAATGGATTTCCTGGAGC
114
174














SNP
Enzyme
Allele 1
Allele 2
Heterozygate














1
AvaI
313, 32, 16, 135
220, 93, 32, 16, 135
313, 220, 135, 93, 32, 16


 


2
PvulI
333
309, 24
333, 309, 24


 


3
TagI
496
315, 181
496, 315, 181


 


4
AvaI
150, 85, 48
150, 85, 32, 16
150, 85, 48, 32, 16


 


5
HincII
249
220, 29
249, 220, 29


 


6
BstNI
215
184, 31
215, 184, 31


 


7
MnlI
240
151, 70, 19
240, 151, 70, 19


 


8
PstI
193
163, 30
193, 163, 30


 


9
BslI
243
219, 22
243, 219, 22


 


10

204
204
204


 


11
DdeI
269
243, 26
269, 243, 26


 


12
BsnFI
162
132, 30
162, 132, 30


 


13
BsmAI
451, 169
401, 169, 50
451, 401, 169, 50


 


14
Mbd
190, 31
221
221, 190, 31


 


15
MnlI
62, 270, 45, 186, 57
62, 12, 258, 45, 186, 57
270, 258, 186, 62, 57, 45, 12


 


16
BstU
225
195, 30
225, 195, 30


 


17
PstI
231
199, 32
231, 199, 32


 


18
HnaI
622
440, 182
622, 440, 182


 


19
MspI
241, 214, 167
181, 60, 214, 167
241, 214, 181, 167, 60


 


20
HnfI
200
176, 24
200, 176, 24


 


21
TagI
146, 389, 87
146, 127, 262, 87
389, 262, 146, 127, 87


 


22
HnaI
331
304, 27
331, 304, 27


 


23
TagI
146, 127, 349
146, 127, 262, 87
349, 262, 146, 127, 87


 


24

259
259
259


 


25
HaeIII
254
96, 158
254, 158, 96


 


26
Acl I
190
160, 30
190, 160, 30


 


27
Bsu 361
254
168, 86
254, 168, 86


 


28
Hinc II
186
158, 28
186, 158, 28


 


29
Alu I
315
252, 63
315, 252, 63


 


30
Bsa H
315
234, 81
315, 234, 81


 


31
EarI
124
80, 44
124, 80, 44


 


32
Ban II
120
80, 38, 2
120, 80, 38, 2


 


33
Ear I
126
94, 32
126, 94, 32


 


34
BsiHKAI
167
102, 65
167, 102, 65


 


35
Sau961
118
89, 29
118, 89, 29


 


36
TfiI
122
67, 55
122, 67, 55


 


37
Ear I
122
95, 27
122, 95, 27


 


38
Eco0109 I
179
151, 28
179, 151, 28


 


39
TspRI
174
141, 33
174, 141, 33
















TABLE 4







Amino Acid Polymorphisms

















Effect on amino acid side


SNP
POSITION
LOCATION
VARIANT 1
VARIANT 2
chain















5
A7164T
EXON 1
MET
LEU
Conservative


6
C10039T
EXON 2
PRO
PRO
Neutral


7
C10082T
EXON 2
LEU
SER
Hydrophobic - Hydrophilic


8
C10108T
EXON 2
GLY
GLY
Neutral


9
G10344A
EXON 2
SER
ASN
Conservative


10
10363
EXON 2
SER insertion
SER deletion
SER insertion/deletion



(AAG)ins


11
A10516G
EXON 2
GLN
GLN
Neutral


12
C10521T
EXON 2
SER
PHE
Hydrophilic - Hydrophobic


13
T10624C
EXON 2
SER
SER
Neutral


14
G10669A
EXON 2
ARG
ARG
Neutral


15
T10873C
EXON 2
TYR
TYR
Neutral


16
G11020A
EXON 2
ALA
ALA
Neutral


17
A11117G
EXON 2
SER
GLY
Hydrophilic - Hydrophobic


18
T11138G
EXON 2
SER
ALA
Hydrophilic - Hydrophobic


19
G11142T
EXON 2
GLY
VAL
Conservative


20
C11145T
EXON 2
SER
LEU
Hydrophilic - Hydrophobic


21
G11233C
EXON 2
SER
SER
Neutral


22
T11260C
EXON 2
CYS
CYS
Neutral


23
G11495A
EXON 2
ASP
ASN
Hydrophilic charged -







Hydrophilic neutral




















TABLE 5





Primer Name
Primer Sequence Forward
SEQ ID NO
Primer Sequence Reverse
SEQ ID NO







SEEK INI_8
CAGTGAGCTGAGACCGTG
115
CTGGTACCAGTGTGTCAG
116


SEEK INI_8
CAGTGAGCTGAGACCGTG
115
CTGGTACCAGTGTGTCAG
116


SEEK INI_8
CAGTGAGCTGAGACCGTG
115
CTGGTACCAGTGTGTCAG
116


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK INI_6
GACTCCTCAGAGCCTCAG
117
GTAGCTACTGAAGCCGCTG
118


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM3
CCTAGATCAAGAGGCCCAG
119
ACAGCAGGAGACTCGAGG
120


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122


SEEK PROM2
CCTCAGATGCTTCATGAATGG
121
GTGAAGTCAGCCGAATAGC
122






















TABLE 6






AC006163
Corneodesmosin
Location





SNP
nt position
nt position
in gene
Flanking Sequence
IUB Code
SEQ ID NO





















1
 6,984 bp
−115
5′UTR
TACCACGCTCCTCCCGGCCACACCAACTTCCCCC/TGGGGCACCCACCCCCTCCACCTCTCCTCCTCTCCC
Y
123


2
 7,068 bp
−31
5′UTR
TGCCCAGGGAATGTCCAGCTCTGGCATAAAGGACCCA/GGGTGTCCTCGAGCTGCCATCAGTCAGGAGGCCG
R
124


3
 7,077 bp
−22
5′UTR
AGCTCTGGCATAAAGGACCCAGGTGTCCTC/TGAGCTGCCATCAGTCAGGAGGCCGTGCAGCCCGAGATGGGC
Y
125


4
 7,107 bp
9
5′UTR
GAGCTGCCATCAGTCAGGAGGCCGTGCAGC/TCCGAGATGGGCTCGTCTCGGGCACCGTGGATGGGGCGT
Y
126


5
 7,164 bp
66
Exon 1
GCACCCTGGATGGGGCGTGTGGGTGGGCACGGGATGA/TTGGCACTGCTGCTGGCTGGTCTCCTCCTGCCAGG
W
127


6
 8,884 bp
Intron 1
Intron 1
CTGGAGGGGCTAGGGAAGGCAGAAGGAACGCAGG T/A GAAAGAGTCATGGAGGAACCATGGGGTAAGTT
W
128


7
 8,906 bp
Intron 1
Intron 1
CAGAAGGAACGCAGGTGAAAGAGTCATGGAGGAACCAT/CGGGGTAAGTTGGGCCTGGGGTTTTGAGCAA
Y
129


8
 8,931 bp
Intron 1
Intron 1
GGAGGAACCATGGGGTAAGTTGGGCCTGGGGTTTTG/CAGCAAAGGAAAGGAAAGATAAGGAAAGATGTGGCTC
S
130


9
 9,538 bp
Intron 1
Intron 1
CTGTCTCTTCAGGGTCCTTTCTTTTAGACCTAT/CTTGTTCCTGCCCCTTCTCCATTCCCTCTTCTTTT
Y
131


10
 9,607 bp
Intron 1
Intron 1
AAAAAAATTTTAATTAAAAAACAAAATACAGAT/CGGGGTCTATGTTGCCCAGGCTGGTCTTGAACTCTGGGGCGC
Y
132


11
 9,608 bp
Intron 1
Intron 1
AAAAAAATTTTAATTAAAAAACAAAATACAGATG/AGGGTCTATGTTGCCCAGGCTCCTCTTGAACTCTGGGGCGC
R
133


12
 9,647 bp
Intron 1
Intron 1
GGGTCTATGTTGCCCAGGCTGGTCTTGAACTCTGGGGCG/ACATGCAATCCTCCCACCTCAGCCTCCCAAAGTGCTGG
R
134


13
 9,667 bp
Intron 1
Intron 1
TCTTGAACTCTGGGGCGCATGCAATCCTCCCACCTCA/GGCCTCCCAAAGTGCTGGGATTACCGGCGTGAGCCACT
R
135


14
 9,745 bp
Intron 1
Intron 1
AGCCCCCTCTTATATTCAATGTATTCCTTTGAGGT/CCACTCACTTTGGCACGTAATTTTCTATTTTTCTGGTTG
Y
136


15
 9,761 bp
Intron 1
Intron 1
TCAATGTATTCCTTTGAGGTCACTCACTTTGGCACG/CTAATTTTCTATTTTTCTGGTTGGTGTTTGCCCACCCTT
S
137


16
 9,926 bp
Intron 1
Intron 1
CCCTGCGCTCTGCTTGGGAGAAACCCGAGAGGCCGATT/GACTGAGATAAGGCAGAAAGGTGAGGGAGGAAGCCA
K
138


17
 9,952 bp
Intron 1
Intron 1
AGAGGCCGATTACTGAGATAAGGCAGAAAGGTGAGGG/AAGGAAGCCAAGCCTCTTTGGCCCTTACTAACCACTG
R
139


18
 9,968 bp
Intron 1
Intron 1
ACTGAGATAAGGCAGAAAGGTGAGGGAGGAAGCCCAAGCCTCT/CTTGGCCCTTACTAACCACTGCTTTCCTCCACAGGGACCTTG
Y
140


19
10,039 bp
137
Exon 2
CAGGGACCTTGGCTAAGAGCATTGGCACCTTCTCAGACCC C/TTGTAAGGACCCCACGCGTATCACCTCCCCTAACGACCCCT
Y
141


20
10,082 bp
180
Exon 2
GGACCCCACGCGTATCACCTCCCCTAACGACCCCTGCC/TTCACTGGGAAGGGTGACTCCAGCGGCT
Y
142


21
10,108 bp
206
Exon 2
ACGACCCCTGCCTCACTGGGAAGGGTGACTCCAGCGGC/TTTCAGTAGCTACAGTGGCTCCAGCAGTTCTGGCAGCTCCAT
Y
143


22
10,344 bp
442
Exon 2
CCGGTTCCTCCCAGCTGGGGAGCAGCAGCTCTCACTCGGGAAG/ACAGCGGCTCTCACTCGGGAAGCAGCAGCTCTCATTCG
R
144


23
10,363 bp (ins)
461
Exon 2
GGAGCAGCAGCTCTCACTCGGGAAGCAGCGGCTCTCACTCGGG(AAG)CAGCAGCTCTCATTCGAGCAGCAGCAGCAGCTT
Ins/del
145


24
10,516 bp
614
Exon 2
AATACTAAACCCTTCCCAGCCTGGACAAAGCTCTTCCTCTTCCCAA/GACCTYTGGGGTATCCAGCAGTGGCCAAAGCGTCAGCTCC
R
146


25
10,521 bp
619
Exon 2
AATACTAAACCCTTCCCAGCCTGGACAAAGCTCTTCCTCTTCCCAAACCTC/TTGGGGTATCCAGCAGTGGCCAAAGCGTCAGCTCC
Y
147


26
103624 bp
722
Exon 2
CGACTCTCCCTGCAGTGGAGGGCCCATCGTCTCGCACTCT/CGGCCCCTACATCCCCAGCTCCCACTCTGTGTC
Y
148


27
10,669 bp
767
Exon 2
CCTACATCCCCAGCTCCCACTCTGTGTCAGGGGGTCAGAGG/ACCTGTGGTGGTGGTGGTGGACCAGCACGGTTCTGGTGC
R
149


28
10,873 bp
971
Exon 2
ACAGTTATCTGGTTCCAGGCATGACCTACAGTAAGGGTAAAATCTAT/CCCTGTGGGCTACTTCACCAAAGAGAACCCTGTGA
Y
150


29
11,020 bp
1118
Exon 2
ACCCCATCATCCCCAGCCAGTCGGCAGCTTCCTCGGCCATTGCG/ATTCCAGCCAGTGGGGACTGGTGGGGTCCAGC
R
151


30
11,117 bp
1215
Exon 2
CCAAGGGACCCTGCTCTCCCTCCAGTTCTCGAGTCCCCAGCA/GGTTCTAGCATTTCCAGCAGCTCCGGTTCACCCTA
R
152


31
11,138 bp
1236
Exon 2
CTCGAGTCCCCAGCAGTTCTAGCATTTCCAGCAGCT/GCCGGTTCACCCTACCATCCCTGCGGCAGTGCTT
K
153


32
11,142 bp
1240
Exon 2
CTAGCATTTCCAGCAGCTCCG G/T TTCACCCTACCATCCCTGCGGCAGTGCT
K
154


33
11,145 bp
1243
Exon 2
CCAGCAGTTCTAGCATTTCCAGCAGCTCCGGTTC/TACCCTACCATCCCTGCGGCAGTGCTTCCCAGAG
Y
155


34
11,233 bp
1331
Exon 2
GGCACCGGGTCCTTCAGCAGCAGCTCCAGTTCCCAATCG/CAGTGGCAAAATCATCCTTCAGCCTTGTGGCAGCAA
S
156


35
11,260 bp
1358
Exon 2
AGTTCCCAATCGAGTGGCAAAATCATCCTTCAGCCTTGT/CGGCAGCAAGTCCAGCTCTTCTGGTCACCCTTGC
Y
157


36
11,495 bp
1593
Exon 2
TGAAGTTTTCCTACCCCAAGGAGAGTTACTCG/AACAGTCCAT(AAG)AAGTCAACTGTTGTGTGTGTGCAT
R
158


37
11,505 bp (ins)
1603
3′UTR
TACCCCAAGGAGAGTTACTCGACAGTCCAT(AAG)AAGTCAACTGTTGTGTGTGTGCATGCCTTGGGCACAAA
Ins/del
159


38
11,575 bp
1674
3′UTR
GGCACAAACAAGCACATACACTATATCCCATATGGGAGAAGG/TCAGTGCCCAGGCATAGGGTTAGCTCAGTTTCCCTCCTTCCCA
K
160


39
11,641 bp
1739
3′UTR
AGCTCAGTTTCCCTCCTTCCCAAAAGAGTGGTTCTGCTTTCTCT/CACTACCCTAAGGTTGCAGACTCTCTCTTATCAC
Y
161


40
11,649 bp
1747
3′UTR
AAAAGAGTGGTTCTGCTTTCTCYACTACCCT/CAAGGTTGCAGACTCTCTCTTATCACCCCTTCCTCCTTCCTC
Y
162


41
11,808 bp
1906
3′UTR
AGATCACCACCCCTTACAATTCCCTCTACTGTGTT/GGAAATGGTCCATTGAGTAACACCCCCATCACCTTCTCAACT
K
163


42
11,839 bp
1937
3′UTR
GAAATGGTCCATTGAGTAACACCCCCATCAC/GCTTCTCAACTGGGAAACCCCTGAAATGCTCTCAGAGCACC
S
164


43
11,885 bp
1963
3′UTR
TGAAATGCTCTCAGAGCACCTCTGA T/C GCCTGAAGAAGTTATACCTTCCTC
Y
165


44
11,977 bp
2075
3′UTR
AACCATCATCTGGAAACAGTGGCCACTTTTCACTGACCTC/TTCTTCGACATCTAGTCAACCCACCCAATATGC
Y
166


45
12,018 bp
2116
3′UTR
ATCTAGTCAACCCACCCAATATGCCACTGGGCTT/CTCGCTCCCAATTCCACCCCACCCTCCATTACAGAGCTCACCA
Y
167


46
12,136 bp
2234
3′UTR
GCCTCTCAAGGCCCTTATCTCAGCCCCTTCCTGTGGCCAT/CTTCCCTCAGTGCCCAGATGATTCCCTGGGTGAGGGCAGACAC
Y
168


47
12,149 bp
2247
3′UTR
CAGCCCCTTCCTGTGGCCATTTCCCTCAGTGCC/TCAGATGATTCCCTGGGTGAGGGAGACACTGGGGCACCCTC
Y
169


48
12,198 bp
2296
3′UTR
TTCCCTGGGTGAGGGAGACACTGGGGCACCCTCAGAGGTTGG/AAGCAGGCTCCCTGCTGTCCCTGGATCCTGGACAGA
R
170


49
12,283 bp
2381
3′UTR
GGTGCAGACTTTTTGCCTTCTTGGA G/A TCCTGGGTCTCCTCTGAGAGTCTG
R
171


50
12,318 bp
2416
3′UTR
TCTTGGAGTCCTGGGTCTCCTCTGAGAGTCTGGGTGGTGCTCT/CTCCTACGCCTCTAGAGGTCTCTGTGTCCCTCA
Y
172


51
12,345 bp
2443
3′UTR
TGGGTGGTGCTCTTCCTACGCCTCTAGAGGTCTCTGTGTC/TCCTCATTTTCCTTCAAAAGCGGGCTGTGTTTCT
Y
173


52
12,373 bp
2471
3′UTR
TCATTTTCCTTCAAAAGCGGGCTGT G/A TTTCTCTTCTACCTTCCAGCTCCT
R
174


53
12,901 bp
2999
3′UTR
TAGATCAAGAGGCCCAGCCTGTGGCAGAACAGAGCTGCCA/GGTGGTCTCTCCATCTTCACACTCCCTGCTCTGCTGGGGT
R
175


54
13,001 bp
3099
3′UTR
AACATGGCTCTCAGGTGAGGGCTGAGAAGGCAGAGTGCCCCA/CGTGGGAAAGAGGAGTCGCTTCCACTGGAGAAGAGAGA
M
176


55
13,020 bp
3118
3′UTR
GCTGAGAAGGCAGAGTGCCCCAGTGGGAAAGAGGAGTCGCT/CTCCACTGGAGAAGAGAGAGAAAGTGGAGTGTGTGGTG
Y
177


56
13,108 bp
3206
3′UTR
GACTTAAGTCCTGAGACAGGCAGGGAGAGGCTGAGGCGGAC/GGAAGTTCCCGCATCCCAAGGAGGGCAGAGTGGATT
S
178


57
13,117 bp
3215
3′UTR
TGAGACAGGCAGGGAGAGGCTGAGGCGGACGAAGTTCCC/TGCATCCCAAGGAGGGCAGAGTGGATTGTGCTTGTCC
Y
179


58
13,178 bp
3276
3′UTR
GGATTGTGCTTGTCCCTGTAGGAGCCCCACCCCCCACCCC/TAGGCCACCTCTCAGAGCCTCTGCTTGGCTGCAAAGG
Y
180


59
13,224 bp
3322
3′UTR
CTCAGAGCCTCTGCTTGGCTGCAAAGGAATTCACCCC/TTACTGTAGCACTTAACCCATTCCCTCCTATCAGGGTGG
Y
181


60
13,316 bp
3414
3′UTR
TGAATTTAGAACTGTTGAAACTCCAAGTCTGGAATCAGCAA/GAAATGTATTACATTGACCAGAAAGGGATTGAATCACCCT
R
182


61
13,365 bp
3463
3′UTR
ACATTGACCAGAAAGGGATTGAATCACCCTTGGTCCAGCA/GTCTGGCCCCTGATCTGCAGCCAATGGCAGGAATCGAGGTC
R
183


62
13,562 bp
3660
3′UTR
AGGCCTCTGGGCTCCATCCACTGCCAGTTCTGGAGA/TGGAGCTCTTCACTCCTCCAGTGGTTAAGCCAGCA
W
184


63
13,605 bp
3703
3′UTR
CTCTTCACTCCTCCAGTGGTTAAGCCAGCAGGGGCAGGT/CGGGGAGGACACAGCAGTAGAATCAGCCAACAGCTCAT
Y
185


64
13,670 bp
3768
3′UTR
CATGTTTAGACCTTGGGCAGCCAGGGAAGCC/TTACTCCTGGGGCCTCCCGGAAGCCATGGAGAGAAC
Y
186


65
13,859 bp
3857
3′UTR
GATCAAGTCCTGGCCATTTGACAGCAGCATTTAAAGGCT/CCTCCTCTACTGTTACTTGGAAATAGCCACTTTCTCCCAAGGT
Y
187


66
13,889 bp
3897
3′UTR
CTCCTCTACTGTTACTTGGAAATAGCCACT/CTTCTCCCAAGGTTTCTTATACTCT
Y
188


67
13,914 bp
3922
3′UTR
GAAATAGCCACTTTCTCCCAAGGTTTCTTATACTCTG/ATGGCACATCTGACCACCAGTAGCAGGCAGAATGATGT
R
189





















TABLE 7








AC006163



Frequency













SNP
nt position
nt position*
SNP
chemistry
allele 1
allele 2
















1
 6,984 bp
44884
CDSN6984
PSQ
69.6
30.4


2
 7,068 bp
44968
CDSN7068
PSQ
60.8
39.2


19
10,039 bp
47939
PS SEEK IN 1 6 C565T
Sequenced
55
45


21
10,108 bp
48008
CDSN C10098T
Sequenced
not available
not available


22
10,344 bp
48244
CDSN G10343A
Sequenced
not available
not available


23
10,363 bp (ins)
48262
CDSN 10363 AAG ins
Sequenced
not available
not available


24
10,516 bp
48416
CDSNx2.2A10516G
PSQ
47.8
52.2


25
10,521 bp
48421
CDSNx2.2C10521T
PSQ
20.5
79.5


26
10,624 bp
48524
CDSNx2T10614C
SNaPshot
48.9
51.1


27
10,669 bp
48569
CDSNx2.2G10669A
SNaPshot
85.7
14.3


28
10,873 bp
48773
CDSN T10873C
SNaPshot
32.3
67.7


29
11,020 bp
48920
SEEKIN1_3 G27A
PSQ
43.8
56.2


30
11,117 bp
49017
SEEKIN1_3 A124G
PSQ
98.8
1.2


31
11,138 bp
49038
SEEKIN1_3 T145G
PSQ
82.8
17.2


32
11,142 bp
49042
SEEKIN1_3 G149T
PSQ
100
0


33
11,145 bp
49045
SEEKIN1_3 C152T
PSQ
64.3
35.7


34
11,233 bp
49133
SEEKIN1_3 G241C
PSQ
47.8
52.2


35
11,260 bp
49160
SEEKIN1_3 T268C
PSQ
78.9
21.1


36
11,495 bp
49395
SEEK1in3 G503A
SNaPshot
68.7
31.3


37
11,505 bp (ins)
49404–49407
SEEK1in3.511INS
SNaPshot
43.4
56.6


38
11,575 bp
49479
CDSN G11576T
SNaPshot
32.5
67.5



























TABLE 8






SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-



1
2
19
21
22
23
24
25
26
27
28


























SNP1
1
0.76
0.89
0.75
−0.88
−0.19
0.55
0.23
−0.56
0.13
−0.47


SNP2
1
0.81
0.74
−0.8
−0.3
0.45
0.18
−0.42
0.17
−0.27
−0.6


SNP19

1
0.79
−0.91
−0.26
0.56
0.19
−0.54
0.15
−0.41
−0.6


SNP21


1
−0.84
−0.3
0.43
0.16
−0.39
0.07
−0.35
−0.48


SNP22



1
0.26
−0.56
−0.18
0.52
−0.13
0.41
0.55


SNP23



1
0.55
0.23
−0.55
0.17
−0.4
−0.47
−0.14


SNP24





1
0.41
−0.99
0.36
−0.71
−1


SNP25






1
−0.43
−0.27
−0.59
−0.33


SNP26







1
−0.33
0.71
1


SNP27








1
0.34
−0.33


SNP28









1
0.65


SNP29










1


SNP30












SNP36













SNP37













SNP38


































SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-
SNP-




29
30
31
32
33
34
35
36
37
37























SNP1
−0.57
−0.1
0.04
n/a
0.46
−0.55
0
−0.92
0.55
0.55



SNP2
0.03
0.16
n/a
0.41
−0.48
0.05
−0.8
0.45
0.29
0.29



SNP19
−0.09
0.12
n/a
0.46
−0.55
0.08
−0.93
0.55
0.42
0.42



SNP21
−0.01
0.05
n/a
0.33
−0.39
0.11
−0.8
0.4
0.34
0.34



SNP22
0.08
−0.11
n/a
−0.45
0.54
−0.06
0.94
−0.53
−0.41
−0.41



SNP23
0.14
n/a
0.52
−0.5
−0.26
0.27
0.54
0.4
0.4



SNP24
−0.16
0.3
n/a
0.86
−0.96
−0.28
−0.56
1
0.71
0.71



SNP25
0.02
−0.29
n/a
0.36
−0.34
−0.25
−0.2
0.43
0.58
0.58



SNP26
0.15
−0.28
n/a
−0.86
0.94
0.22
0.55
−0.98
−0.71
−0.71



SNP27
0.08
0.93
n/a
0.27
−0.31
0
−0.16
0.33
−0.36
−0.36



SNP28
0.18
0.35
n/a
−0.64
0.67
0.25
0.44
−0.71
−0.97
−0.97



SNP29
0.02
−0.14
n/a
−0.67
1
−0.34
0.61
−1
−0.72
−0.72



SNP30
1
0.1
n/a
−0.15
0.13
−0.02
0.11
−0.13
−0.18
−0.18



SNP31

1
n/a
0.3
−0.25
−0.01
−0.13
0.27
−0.37
−0.37



SNP32



n/a
n/a
n/a
n/a
n/a
n/a
n/a



SNP33



1
−0.82
−0.25
−0.48
0.86
0.64
0.64



SNP34




1
0.18
0.58
−0.95
−0.68
−0.68



SNP35





1
−0.1
−0.23
−0.23
−0.23



SNP36






1
−0.55
−0.44
−0.44



SNP37







1
0.72
0.72



SNP38








1
1
























TABLE 9














Number of Transmissions















SNP


Frequency

chi-
p value
Allele 1
Allele 2

















Number
Position
SNP Type
(allele 1)
Transmissions
squared
(bootstrap)
observed
expected
observed
expected




















1
44884
Promotor
0.79
117
3.99
0.043
235
226
53
62


2
44968
Promotor
0.69
98
1.14
0.213
157
152
67
72


19
47939
Silent
0.79
133
11.43
0.002
260
244
50
66


21
48008
leu-ser
0.74
125
10.7
0
238
221
60
77


22
48244
ser-asn
0.2
132
3.44
0.061
55
64
259
250


23
48262
ins/del (ser)
0.82
112
9.28
0
231
219
33
45


24
48416
Silent
0.59
125
18.03
0
203
180
99
122


25
48421
ser-phe
0.18
120
1.43
0.18
44
49
232
227


26
48524
silent
0.43
140
22.93
0
113
143
229
199


27
48569
silent
0.13
139
5.97
0.025
35
45
305
295


28
48773
silent
0.56
142
36.51
0
152
188
194
158


29
48920
silent
0.47
26
0.99
0.283
23
26
33
31


30
49017
ser-gly
0.96
131
11.16
0
291
299
21
13


31
49038
ser-ala
0.13
135
4.55
0.051
34
42
290
282


32
49042
gly-val
1


33
49045
ser-leu
0.59
132
9.69
0
211
193
111
129


34
49133
ser-leu
0.43
133
11
0.002
115
135
203
183


35
49160
silent
0.33
102
0.74
0.381
77
81
169
165


36
49395
silent
0.22
140
6.47
0.02
61
74
281
268


37
49404
ins/del
0.58
139
18.32
0
223
197
113
139


38
49479
3′ UTR
0.44
144
34.99
0
194
158
156
192




















TABLE 10a









SNP
Haplotype













Number
A
B
C







 1
1
1
2



 2
1
1
2



19
1
1
2



21
1
1
2



22
2
2
1



23
1
1
1



24
1
1
2



25
1
2
2



26
2
2
1



27
2
2
2



28
2
2
1



30
1
1
1



31
2
2
2



33
1
1
2



34
2
2
1



35
2
2
1



36
2
2
1



37
1
1
2



38
1
1
2





















TABLE 10b









Key
Code












Key
1
2







A/T
A
T



A/G
A
G



A/C
A
C



C/G
G
C



G/T
G
T



C/T
C
T

## Claims

1. A recombinant or isolated polynucleotide comprising the corneodesmosin gene of (SEQ ID NO:1), wherein said corneodesmosin gene comprises a nucleotide substitution at position 3382 of SEQ ID NO:1.

2. A vector comprising the polynucleotide according to any one of claim 1.

3. An isolated host cell comprising the polynucleotide as claimed in claim 1; or
an isolated host cell comprising a vector wherein said vector comprising the polynucleotide as claimed in claim 1.

4. A recombinant or isolated polynucletide wherein said nucleic acid sequence has full complementarity to the polynucleotide as claimed in claim 1.